Curvature induced membrane structure from fluorescence

Intrinsic curvature difference between lipids drive lipid organization in mixed lipid membranes. Steady state fluorescence of bilayer soluble Laurdan and water soluble Fluorescein were able to elucidate the packing heterogeneity and composition dependent shape change of mixed lipid vesicles. Mixtures included gel and liquid phase lipids. Computed second harmonic (SH) spectra of Laurdan, in all cases, revealed two lines. Spectra were fit to two log-normal distributions. The SH of Fluorescein showed three peaks. Areas, peak intensities, positions, and linewidths of the resolved lines were examined as functions of lipid mixture composition. Area of the Laurdan blue line from the more packed lower curvature lower polarity regions, Ab, increased and that of the red line from less packed higher curvature higher polarity regions, Ar, decreased with the higher intrinsic curvature lipid fraction. The red peak shifted to longer wavelengths but the blue peak did not. Area changes indicate increased presence of Laurdan in packed flatter regions. Red-shift in red peak position indicates decrease in packing and increase in polarity of the region of origin of the red emission. Fluorescein being outside the vesicle does not sense changes within the bilayer but line parameters showed evidence of change in vesicle shape due to scattering of the fluorescence. These results are consistent with the model of separation of lipids of dissimilar intrinsic curvatures into domains differing in packing and increase in the extent of the flatter sections to relieve curvature stresses. Growth in packed flat regions together with Laurdan's preference for lower curvature regions accounts for the increase in blue and decrease in red emissions. The added higher curvature lipid enters the higher curvature part of the bilayer and causes a red-shift of the peak from this region, but does not affect the blue peak position.